Vesicles were first described in the 1960s as a model of cellular membranes (see Bangham et al., J. Mol. Biol. 13:238-252, 1965). Vesicles have found a number of applications in the delivery of small molecule drugs, vaccine adjuvancy, gene transfer and diagnostic imaging (e.g., see Liposome Technology, 3rd Edition, Edited by Gregory Gregoriadis, Informa HealthCare, 2006 and Liposomes: A Practical Approach (The Practical Approach Series, 264), 2nd Edition, Edited by Vladimir Torchilin and Volkmar Weissig, Oxford University Press, USA, 2003).
A number of methods for preparing vesicles have been described (e.g., see references cited above and Walde and Ichikawa, Biomol. Eng., 18:143-177, 2001). However, there remains a need in the art for methods that can be used to entrap substances within vesicles. One method for entrapping small molecules was originally described in 1995 which employed a tert-butanol and water co-solvent system (see Kasrian and DeLuca, Pharm. Res., 12:484-490, 1995 and Kasrian and DeLuca, Pharm. Res., 12:491-495, 1995). Specifically, the method involves dissolving the lipids (and any other organic solvent soluble materials) in tert-butanol and dissolving any water-soluble materials such as sucrose in water. These two solutions are then mixed in an appropriate ratio to produce a third monophase solution. The resulting solution is freeze-dried to form a lyophilized product. The lyophilized product is then reconstituted by the addition of an equal volume of water and gentle shaking, which leads to the formation of an aqueous suspension of vesicles. This method of vesicle preparation has been used to entrap small molecule drugs (e.g., see Li and Deng, J. Pharm. Sci. 93:1403-1414, 2004 and Alexopoulou et al., J. Liposome Res. 16:17-25, 2006). As described by Li and Deng, this has been achieved by either including the small molecule drug in the initial monophase solution (passive loading) or using pH gradients to load the small molecule drug into empty pre-formed vesicles (active loading).
While these methods may well be suitable for entrapping substances that can withstand contact with organic solvents such as tert-butanol and/or small molecules that are able to diffuse rapidly into empty vesicles we have found it unsuitable for entrapping the types of antigens (e.g., polypeptides, viruses, etc.) that are commonly involved in vaccines. In particular, we have found that these methods produce low entrapment efficiencies and can dramatically reduce the activity of the underlying antigen (e.g., as measured by immune responses). There is therefore a need in the art for methods of preparing vesicles that are capable of entrapping antigens while minimizing impact on antigen activity.